Supplemental Information. Systematic Revision of Symbiodiniaceae Highlights. the Antiquity and Diversity of Coral Endosymbionts

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1 Current Biology, Volume 28 Supplemental Information Systematic Revision of Symbiodiniaceae Highlights the Antiquity and Diversity of Coral Endosymbionts Todd C. LaJeunesse, John Everett Parkinson, Paul W. Gabrielson, Hae Jin Jeong, James Davis Reimer, Christian R. Voolstra, and Scott R. Santos

2 Figure S1. Molecular clock calibrations for Symbiodiniaceae. Relates to Figures 1, S2, S3, and S4, and to STAR Methods Section 4: Quantification and Statistical Analysis. Calibrations using paleontological evidence, plate-tectonic events, and biogeographic data to establish an rdna-based molecular clock for estimating times of origination and divergence among Symbiodiniaceae clades.

3 Figure S2. Calibration times for the radiation of symbiotic dinoflagellates specialized for soritid foraminifera. Relates to Figures 1, S1, S3, and S4, and to STAR Methods Section 4: Quantification and Statistical Analysis. (A) The major adaptive radiation of large foraminifera, including Soritoidea, occurred during the Eocene Climactic Optimum. Timing of the Paleocene Eocene Thermal Maximum (PETM) is indicated. (B) The Eocene occurrence and ecology of Orbitolites, the adaptive radiation of Soritoidea, and new molecular clock estimates for Sorites place the most likely time of origin for these symbiont lineages in the early Eocene. Solid lines are based on paleontological evidence, which defines the latest possible time of origin. The dashed portion of each line indicates earlier times of emergence likely predate estimates based on available fossil evidence. Times of origination and extinctions (cross symbol) were obtained from BouDagher-Fadel [S1].

4 Figure S3. Proposed calibration time of two distantly related lineages previously grouped together and subjectively designated as Clade D based on RFLP digestions of small subunit ribosomal DNA (SSU rdna). Relates to Figures 1, S1, S2, and S4, and to STAR Methods Section 4: Quantification and Statistical Analysis. One lineage is common to various cnidarians, including stony and soft corals, and is known for exhibiting high thermal tolerances. The other lineage is specific to soritid foraminifera and hence designated here as Foraminifera Clade D.

5 Figure S4. Phylogeny of the diversity of Clade H from the Pacific and Atlantic Oceans inferred by Maximum Parsimony (MP). Relates to Figures 1, S1, S2, and S3, and to STAR Methods Section 4: Quantification and Statistical Analysis. Sequences used by Pochon et al. [S2] are specified and contrasted with sequences identified to be more appropriate for molecular clock calibrations based on the geological separation of Pacific and Atlantic Ocean biota.

6 Cell Length (µm) Cell Width (µm) Sym. Genus* Species Ref. N Mean SD Mean SD Clade A Symbiodinium linucheae DM A Symbiodinium microadriaticum [S3] A Symbiodinium natans DM A Symbiodinium necroappetens [S4] A Symbiodinium pilosum DM A Symbiodinium tridacnidorum [S3] B Symbiodinium aenigmaticum [S5] B Symbiodinium antillogorgium [S5] B Symbiodinium endomadracis [S5] B Symbiodinium minutum [S6] B Symbiodinium pseudominutum [S5] B Symbiodinium psygmophilum [S6] C Symbiodinium goreaui DM C Symbiodinium thermophilum DM D Symbiodinium boreum [S7] D Symbiodinium eurythalpos [S7] D Symbiodinium glynnii [S8] D Symbiodinium trenchii [S7] E Symbiodinium voratum [S9] F Symbiodinium kawagutii DM G Symbiodinium endoclionum [S10] G Symbiodinium spongiolum [S10] 1 NA N/A Ansanella granifera [S11] 2 NA N/A Asulcocephalium miricentonis [S12] 2 NA N/A Biecheleria baltica [S13] 2,3 NA N/A Biecheleria brevisulcata [S14] 2,3 NA N/A Biecheleria cincta [S15] 2 NA N/A Biecheleria halophila [S16] 3 NA N/A Biecheleria natalensis [S17] 2,3 NA N/A Biecheleria pseudopalustris [S18] 2,3 NA N/A Biecheleriopsis adriatica [S19] NA N/A Leiocephalium pseudosanguineum [S12] 2 NA N/A Pelagodinium beii [S20] NA N/A Polarella gracialis [S21] 2 NA N/A Protodinium corii [S15] 2 NA N/A Protodinium simplex [S15] 2 NA N/A Yihiella yeosuensis [S22] NA * Genus Symbiodinium to be retained only for Clade A. 1 Supplemented with additional measurements that may not have appeared in the original publication. 2 Sizes were reported as ranges, mean was inferred as the average of the extremes and SD as half of the range. 3 Only length was reported, width was inferred to be identical. Table S1. Suessiales cell size measurements. Relates to Figure 2 and to STAR Methods Section 3: Method Details. Morphological measurements (cell length and width) for each Symbiodiniaceae species, and other related dinoflagellates, included in this study. N = number of independent in vitro cultures or in hospite tissue samples measured (>40 cells each); DM = direct measurement; SD = standard deviation; NA = not applicable.

7 Clade A (Symbiodinium) Clade A (Temperate) Clade B (Breviolum) Clade C (Cladocopium) Clade D (Durusdinium) Clade A (Symbiodinium) 0.03 Clade A (Temperate) Clade B (Breviolum) Clade C (Cladocopium) Clade D (Durusdinium) Clade D (Foraminifera) Clade E (Effrenium) Clade Fr Clade Fr Clade Fr Clade Fr5 (Fugacium) Clade G (Foraminifera) Clade G (Gerakladium) Clade H Clade I Clade D (Foraminifera) Clade E (Effrenium) Clade Fr2 Clade Fr3 Clade Fr4 Clade Fr5 (Fugacium) Clade G (Foraminifera) Clade G (Gerakladium) Clade H Clade I Table S2. Genetic distances of LSU rdna between Symbiodiniaceae. Relates to Figures 3A and 4 and to STAR Methods Section 4: Quantification and Statistical Analysis. Corrected pairwise genetic distances among LSU rdna sequences from new genera and genus-level groups of Symbiodiniaceae. Values were generated under the General Time Reversible model of evolution, with a proportion of invariable sites and rate variation among sites (i.e. GTR+I+G) estimated from the data, and averaged over all sequences within each genus/group. Shaded values on the diagonal represent the within-genus divergence among species. Estimates <0.01 were rounded up to this minimum value.

8 Clade A (Symbiodinium) Clade B (Breviolum) Clade C (Cladocopium) Clade D (Durusdinium) Clade E (Effrenium) Clade Fr5 (Fugacium) Clade G (Gerakladium) Clade A (Symbiodinium) 0.01 Clade B (Breviolum) Clade C (Cladocopium) Clade D (Durusdinium) Clade E (Effrenium) NA Clade Fr5 (Fugacium) Clade G (Gerakladium) Table S3. Genetic distances of mitochondrial cob between Symbiodiniaceae. Relates to Figure 3B and to STAR Methods Section 4: Quantification and Statistical Analysis. Corrected pairwise genetic distances among mitochondrial cob sequences from new genera and genus-level groups of Symbiodiniaceae. Values were generated under the General Time Reversible model of evolution, with a proportion of invariable sites and rate variation among sites (i.e. GTR+I+G) estimated from the data, and averaged over all sequences within each genus/group. Shaded values on the diagonal represent the within-genus divergence among species. Estimates <0.01 were rounded up to this minimum value. NA = not applicable (for genera represented by one species).

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